Microbial production of short and medium chain esters: Enzymes, pathways, and applications

Kruis, Aleksander J.; Bohnenkamp, Anna Christina; Patinios, Constantinos; Nuland, Youri M. van; Levisson, Mark; Mars, Astrid E.; Berg, C. van den; Kengen, Servé W. M.; Weusthuis, Ruud A.

doi:10.1016/j.biotechadv.2019.06.006

Cited by 94 publications

(93 citation statements)

References 218 publications

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“…Ethyl acetate is used on a large scale as an industrial solvent for the production of paints, coatings and resins [30], as well as in the flavours and fragrances industry [9,19]. The global production of the ester was estimated at 3.5 million tonnes in 2015 [42].…”

Section: Introductionmentioning

confidence: 99%

Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Bohnenkamp

Kruis

Mars

et al. 2020

Biotechnol Biofuels

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Background: Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results: We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion: Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

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Section: Introductionmentioning

confidence: 99%

Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Bohnenkamp

Kruis

Mars

et al. 2020

Biotechnol Biofuels

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“…Therefore, we hypothesize that other bottlenecks need to be tackled for more efficient ester production. One critical bottleneck is the availability of an efficient thermostable AAT used to construct the ester production module (Kruis et al, 2019; Layton & Trinh, 2014; Lee & Trinh, 2019; Rodriguez et al, 2014; Tai et al, 2015). Unfortunately, there is not a functional and thermostable AAT identified from a thermophile.…”

Section: Resultsmentioning

confidence: 99%

“…Medium‐chain (C 6 –C 10 ) esters are potential drop‐in biofuels and valuable chemicals with broad industrial applications such as food additives, fragrances, and solvents (Kruis et al, 2019; Lange et al, 2010). Isobutyl acetate, for instance, is a biodegradable solvent that can substitute for methyl isobutyl ketone and toluene in many formulations.…”

Section: Introductionmentioning

confidence: 99%

“…Microbial ester production has recently emerged as an alternative promising platform for eco‐friendly biosynthesis of esters from renewable and sustainable feedstocks (Kruis et al, 2019; Layton & Trinh, 2014; Park, Shaffer, & Bennett, 2009; Rodriguez, Tashiro, & Atsumi, 2014). The ester biosynthesis pathways can utilize either lipase (van den Berg, Heeres, van der Wielen, & Straathof, 2013) or alcohol acetyltransferases (AATs; Layton & Trinh, 2014; Rodriguez et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…The latter pathway is thermodynamically favorable in aqueous fermentation conditions because water is not generated as a byproduct, and the stored energy from the thioester bond of acyl‐CoA helps the effective transfer of an acyl group. This thermodynamic advantage of the AAT‐dependent pathway makes it attractive for ester biosynthesis in microorganisms (Kruis et al, 2019; Layton & Trinh, 2014; Park et al, 2009; Rodriguez et al, 2014; Tai, Xiong, & Zhang, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Seo

Nicely

Trinh

2020

Biotech & Bioengineering

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Medium‐chain esters are versatile chemicals with broad applications as flavors, fragrances, solvents, and potential drop‐in biofuels. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either lipases or alcohol acyltransferase (AAT), but the AAT‐dependent pathway is more thermodynamically favorable in an aqueous fermentation environment. Even though a cellulolytic thermophile Clostridium thermocellum harboring an AAT‐dependent pathway has recently been engineered for direct conversion of lignocellulosic biomass into esters, the production is not efficient. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. The challenge is to identify and disrupt CEs that can alleviate ester degradation while not negatively affecting the efficient and robust capability of C. thermocellum for lignocellulosic biomass deconstruction. In this study, by using bioinformatics, comparative genomics, and enzymatic analysis to screen a library of CEs, we identified and disrupted the two most critical CEs, Clo1313_0613 and Clo1313_0693, that significantly contribute to isobutyl acetate degradation in C. thermocellum. We demonstrated that an engineered esterase‐deficient C. thermocellum strain not only reduced ester hydrolysis but also improved isobutyl acetate production while maintaining effective cellulose assimilation.

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Multivariate Silicification‐Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity–Stability Trade‐Off

Zheng,

Yang,

Zhou

et al. 2024

Angewandte Chemie

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The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long‐term stability is highly desired but highly challenging. In this work, we propose a new concept of silica‐enzyme, referred to as “silicazyme”, where solid inorganic silica was controlled hybridization with fragile enzyme under moderate condition at single‐enzyme level, realizing simultaneous structure augmentation, long‐term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity‐stability trade‐off the structure flexibility/rigidity of silicazyme was optimized by a component‐adjustment‐ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes that promise to broaden their utilization in catalysis, separations, and nanomedicine.

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Microbial production of short and medium chain esters: Enzymes, pathways, and applications

Cited by 94 publications

References 218 publications

Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Multivariate Silicification‐Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity–Stability Trade‐Off

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